Infection of Common Marmosets with GB Virus B Chimeric Virus Encoding the Major Nonstructural Proteins NS2 to NS4A of Hepatitis C Virus.

UNLABELLED: A lack of immunocompetent-small-primate models has been an obstacle for developing hepatitis C virus (HCV) vaccines and affordable antiviral drugs. In this study, HCV/GB virus B (GBV-B) chimeric virus carrying the major nonstructural proteins NS2 to NS4A (HCV NS2 to -4A chimera) was produced and used to infect common marmosets, since HCV NS2 to NS4A proteins are critical proteases and major antigens. Seven marmosets were inoculated intrahepatically with HCV NS2 to -4A chimera RNA for primary infection or intravenously injected with chimera-containing serum for passage infection. Three animals used as controls were injected with phosphate-buffered saline (PBS) or GBV-B, respectively. Six of seven HCV NS2 to -4A chimera-infected marmosets exhibited consistent viremia and one showed transient viremia during the course of follow-up detection. All six infected animals with persistent circulating viremia presented characteristics typical of viral hepatitis, including viral RNA and proteins in hepatocytes and histopathological changes in liver tissue. Viremia was consistently detected for 5 to 54 weeks of follow-up. FK506 immunosuppression facilitated the establishment of persistent chimera infection in marmosets. An animal with chimera infection spontaneously cleared the virus in blood 7 weeks following the first inoculation, but viral-RNA persistence, low-level viral protein, and mild necroinflammation remained in liver tissue. The specific antibody and T-cell response to HCV NS3 in this viremia-resolved marmoset was boosted by rechallenging, but no viremia was detected during 57 weeks of follow-up. The chimera-infected marmosets described can be used as a suitable small-primate animal model for studying novel antiviral drugs and T-cell-based vaccines against HCV infection. IMPORTANCE: HCV infection causes approximately 70% of chronic hepatitis and is frequently associated with primary liver cancer globally. Chimpanzees have been used as a reliable primate model for HCV infection, but ethical considerations have restricted their utility in biomedical research. GB virus B (GBV-B) is a flavivirus related to HCV. It can infect common marmosets, a New World small primate, and induces viral hepatitis similar to HCV infection in humans. To minimize differences between GBV-B and HCV, we generated HCV NS2 to -4A/GBV-B chimeric viruses and established a chimera-infected marmoset model. HCV NS2 to -4A chimera-infected marmosets provide a small-animal model for evaluating novel antiviral drugs targeting HCV NS3-NS4A protease and T-cell-based HCV vaccines.

A chimeric GB virus B encoding the hepatitis C virus hypervariable region 1 is infectious in vivo.

Two GB virus B (GBV-B) chimeric genomes, GBV-HVR and GBV-HVRh (with a hinge), containing the coding region of the immunodominant hypervariable region 1 (HVR1) of the E2 envelope protein of Hepatitis C virus (HCV) were constructed. Immunoblot analysis confirmed that HVR1 was anchored to the GBV-B E2 protein. To investigate the replication competence and in vivo stability of in vitro-generated chimeric RNA transcripts, two naïve marmosets were inoculated intrahepatically with the transcripts. The GBV-HVR chimeric genome was detectable for 2 weeks post-inoculation (p.i.), whereas GBV-HVRh reverted to wild type 1 week p.i. Sequencing analysis of the HVR1 and flanking regions from GBV-HVR RNA isolated from marmoset serum demonstrated that the HVR1 insert remained unaltered in the GBV-HVR chimera for 2 weeks. Inoculation of a naïve marmoset with serum collected at 1 week p.i. also resulted in viraemia and confirmed that the serum contained infectious particles. All animals cleared the infection by 3 weeks p.i. and remained negative for the remaining weeks. The chimera may prove useful for the in vivo examination of any HCV HVR1-based vaccine candidates.

Core protein domains involved in hepatitis C virus-like particle assembly and budding at the endoplasmic reticulum membrane.

Hepatitis C virus (HCV) core protein, expressed with a Semliki forest virus (SFV) replicon, self-assembles into HCV-like particles (HCV-LPs) at the endoplasmic reticulum (ER) membrane, providing an opportunity to study HCV particle morphogenesis by electron microscopy. Various mutated HCV core proteins with engineered internal deletions were expressed with this system, to identify core domains required or dispensable for HCV-LP assembly. The HCV core protein sequence was compared with its counterpart in GB virus B (GBV-B), the virus most closely related to HCV, to identify conserved domains. GBV-B and HCV display similar tropism for liver hepatocytes and their core proteins are organized similarly into three main domains (I, II and III), although GBV-B core is smaller and lacks approximately 35 amino acids (aa) in domain I. The deletion of short hydrophobic domains (aa 133-152 and 153-167 in HCV core) that appear highly conserved in domain II of both GBV-B and HCV core proteins resulted in loss of HCV core ER anchoring and self-assembly into HCV-LPs. The deletion of short domains found within domain I of HCV core protein but not in the corresponding domain of GBV-B core according to sequence alignment had contrasting effects. Amino acids 15-28 and 60-66 were shown to be dispensable for HCV-LP assembly and morphogenesis, whereas aa 88-106 were required for this process. The production of GBV-B core protein from a recombinant SFV vector was associated with specific ER ultrastructural changes, but did not lead to the morphogenesis of GBV-B-LPs, suggesting that different budding mechanisms occur in members of the Flaviviridae family.

A chimeric GB virus B with 5' nontranslated RNA sequence from hepatitis C virus causes hepatitis in tamarins.

Only humans and chimpanzees are fully permissive for replication of hepatitis C virus (HCV), an important cause of liver cirrhosis and cancer worldwide. The absence of suitable animal models limits opportunities for in vivo evaluation of candidate hepatitis C therapeutics and slows progress in the field. Here, we describe a chimeric virus derived from GB virus B (GBV-B), an unclassified hepatotropic member of the family Flaviviridae that is closely related to HCV and infects tamarins (Saguinus sp.), in which a functionally important HCV regulatory sequence replaced an analogous sequence in the 5' nontranslated region (5'NTR) of the GBV-B genome. The transplanted sequence comprised domain III of the internal ribosome entry site (IRES), which directly binds the 40S ribosome subunit and is a target for candidate therapeutics. The chimeric 5'NTR retained ribosome binding activity and was competent in directing protein translation both in cell-free translation reactions and in transfected primary tamarin hepatocyte cultures. Virus rescued from the chimeric RNA replicated in the liver of tamarins, causing biochemical and histopathological changes typical of viral hepatitis. However, adaptive mutations were required elsewhere in the genome for efficient replication. Virus was not rescued from other, translationally competent, chimeric RNAs in which domain II of the IRES was exchanged. Thus, the 5'NTR appears to contain virus-specific replication signals that interact with other sites within the viral genome or with viral proteins. In conclusion, such novel chimeric flaviviruses offer opportunities for new insights into HCV replication mechanisms, while potentially facilitating the evaluation of candidate therapeutics in vivo.